This patent application is related to U.S. patent application Ser. No. 08/077,214, filed on even date and having a common title herewith.
Zone electrophoresis in capillaries is widely used to accomplish liquid-phase separations of various solutes. Capillary electrophoresis has been used for separation of small and large molecules, various amino acids, alkylamines and various proteins. In brief, a zone capillary electrophoresis device includes a buffer filled capillary tube that is placed between two buffer reservoirs. A potential field is applied across the length of the capillary tube and ionic solutes in one buffer reservoir then differentially migrate through the capillary into the other reservoir. Small diameter silica based tubes are employed as the capillaries in capillary zone electrophoresis (CZE) instruments.
A distinguishing property of flow through a capillary is electroosmotic flow. Immediately adjacent to the solid-liquid interface at the interior of the silica-based capillary wall, a stagnant double layer of solute/solvent is found. Under normal aqueous conditions, the silica capillary wall surface has an excess of charge resulting from an ionization of surface functional groups. Thus, SiOH groups are ionized leaving SiO.sup.- at the wall surface and H.sup.+ ions in the solution and in the stagnant double layer adjacent to the capillary wall. This action creates a potential across the layers which is termed the zeta potential. The zeta potential is dependent upon the viscosity of the fluid, the dielectric constant of the solution and the coefficient of electroosmotic flow of the solution. The cationic counter ions (H.sup.+) in the diffuse solvent/solute layer migrate towards the cathode and because these ions are solvated, they drag solvent with them. The extent of the potential drop across the double layer governs the rate of flow. It is known that control of electroosmotic flow is effective in improving electrophoretic resolution and efficiency and is a controlling factor in obtaining reproducible results in CZE apparatus.
The prior art evidences a number of ways to alter electroosmotic flow. Hjerten indicates that inner surfaces of a capillary can be derivatized by coating them with a mono-molecular layer of non-cross-linked polyacrylamide. This coating encourages the osmotic effect and discourages adsorption of solutes onto the inside of the capillary. See "High Performance Electrophoresis, Elimination of Electroendosmosis and Solute Adsorption", Hjerten, Journal of Chromatography, 347 (1985), pp. 191-198. Others have taught that electroosmotic flow may be altered by altering the buffer pH, the concentration of the buffer, the addition of surface-active species such as surfactants, glycerol, etc. or various organic modifiers to the buffer solution.
For additional details regarding capillary electrophoresis instrumentation and methods of control of electrophoretic separation, the following papers provide a useful oversight of the field: "Capillary Electrophoresis", Ewing et al., Analytical Chemistry, 1989, Volume 61, 292A and "Capillary Electrophoresis", Kuhr, Analytical Chemistry, 1990, Volume 62, 403R-414R.
Independent control of electroosmotic flow (i.e., not related to changes in the buffer or inner capillary structures) have been accomplished by application of external electric fields. As indicated above, it is known that separation resolution can be enhanced and protein adsorption prevented by dynamically controlling the polarity and magnitude of the zeta potential at the boundary between the aqueous fluid and the capillary wall. Lee et al. in "Direct Control of the Electroosmosis in Capillary Zone Electrophoresis by Using an External Electric Field", Analytical Chemistry, 1990, Volume 62, pp. 1550-1552, employ an additional electric field from outside the capillary to enable external control of the zeta potential. Lee et al. mounted a capillary tube inside another tube, filled the annular space therebetween with a potassium phosphate buffer and applied high voltage across the annular space to achieve an electric field along the entire length of the capillary. A pump was used to provide fluid flow of the potassium phosphate buffer to enhance transfer of the heat created by current flow through the buffer. It was determined that by varying the electric field, changes in both the direction and flow rate of electroosmosis in the inner capillary could be achieved. In U.S. Pat. No. 5,151,164, to Blanchard and Lee, the concepts disclosed in the aforementioned Lee et al. article are expanded to include a non-aqueous conductive member surrounding capillary along its entire length.
Hayes et al., "Analytical Chem.", Vol. 64, (1992) pp. 512-516, have achieved a similar control of electroosmotic flow by the application of a radial voltage field about the length of the capillary, but avoided the necessity for an annular fluid flow region as taught by Lee et al. Hayes et al. coated the exterior surface of a capillary with a flexible conductive polymer sheath and then applied a voltage thereacross to achieve a radial field effect within the capillary. Both Hayes et al. and Lee et al. teach the control of electroosmotic flow via an applied radial voltage field and depend upon an application of a radial voltage to all or nearly all of the length of the electrophoresis capillary. Furthermore, all employ a current flow in the material surrounding the capillary to achieve the radial electric field. Such current flows create additional Joule heating within the capillary which adds to the Joule heating that occurs as a result of current flow through the buffer (caused by the voltage applied across the capillary to achieve electrophoretic flow). As is also apparent from the prior art, all flow control fields taught in the prior art were of the electrodynamic variety wherein the fields were created by a current flow in a conductive media adjacent the capillary.
Accordingly, it is an object of this invention to provide an improved system for controlling electroosmotic flow in CZE apparatus.
It is another object of this invention to provide an electroosmosis control structure for a CZE apparatus which is readily manufacturable.
It is yet another object of this invention to provide an electroosmotic flow control system for CZE apparatus which avoids creation of unnecessary Joule heating of the capillary.